Single-chain cd40-receptor agonist proteins

Abstract

Provided herein are specific CD40 receptor agonist proteins, nucleic acids encoding the same, and methods of treating a subject having a CD40L-associated disease or disorder. The CD40 receptor agonist proteins provided herein comprise three soluble CD40L domains and an Fc fragment. The CD40 receptor agonist proteins are substantially non-aggregating and suitable for therapeutic, diagnostic and/or research applications.

1 . A CD40 receptor agonist protein comprising a single-chain fusion polypeptide comprising:
 (i) a first soluble CD40L domain,   (ii) a first peptide linker,   (iii) a second soluble CD40L domain,   (iv) a second peptide linker, and   (v) a third soluble CD40L domain, and   (vi) an antibody Fc fragment, comprising the amino acid sequence as shown in SEQ ID NO: 13 or 14 or amino acids 1-217 of SEQ ID NO: 13,
 wherein the soluble CD40L domains (i), (iii) and (v) each consists of amino acids 120-261 or 121-261 of SEQ ID NO: 1, and the antibody Fc fragment (vi) is located C-terminally to the third CD40L domain (v), and is fused to the soluble CD40L domain (v) via a hinge-linker of any one of SEQ ID NOs: 16 and 19-24. 
   
     
     
         2 . The CD40 receptor agonist protein of  claim 1 , wherein the soluble CD40L domains (i), (iii) and (v) consist of amino acids 121-261 of human CD40L of SEQ ID NO: 1. 
     
     
         3 . The CD40 receptor agonist protein of  claim 1 , wherein the first and second peptide linkers (ii) and (iv) independently have a length of 3-8 amino acids. 
     
     
         4 . The CD40 receptor agonist protein of  claim 3 , wherein the first and second peptide linkers (ii) and (iv) independently consist of glycine and serine, or consist of glycine, serine and asparagine, wherein the asparagine is optionally glycosylated. 
     
     
         5 . The CD40 receptor agonist protein of  claim 1 , further comprising an N-terminal signal peptide domain. 
     
     
         6 . The CD40 receptor agonist protein of  claim 1 , comprising the amino acid sequence of SEQ ID NO: 26, 29, or 30. 
     
     
         7 . The CD40 receptor agonist protein of  claim 1 , comprising two polypeptides each having the amino acid sequence as set forth in SEQ ID NO: 26, 29, or 30. 
     
     
         8 . The CD40 receptor agonist protein of  claim 7 , wherein the two polypeptides are covalently linked through three interchain disulfide bonds formed between cysteine residues 453, 459 and 462 of each polypeptide. 
     
     
         9 . The CD40 receptor agonist protein of  claim 7 , wherein one or more of the asparagine residues at positions 147 and 296 of the polypeptides are N-glycosylated. 
     
     
         10 . The CD40 receptor agonist protein of  claim 1 , wherein the polypeptide is further post-translationally modified. 
     
     
         11 . The CD40 receptor agonist protein of  claim 10 , wherein the post-translational modification comprises modification of the N-terminal glutamine to pyroglutamate. 
     
     
         12 . A pharmaceutical or diagnostic composition comprising the CD40 receptor agonist protein of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         13 . A CD40 receptor agonist protein comprising a single-chain fusion polypeptide comprising:
 (i) a first soluble CD40L domain,   (ii) a first peptide linker,   (iii) a second soluble CD40L domain,   (iv) a second peptide linker, and   (v) a third soluble CD40L domain, and   (vi) an antibody Fc fragment, wherein the antibody Fc fragment (vi) consists of the amino acid sequence of SEQ ID NO: 13 or 14 or amino acids 1-217 of SEQ ID NO: 13;   wherein the soluble CD40L domains (i), (iii) and (v) each consists of amino acids 120-261 or 121-261 of SEQ ID NO: 1, with one or two mutations selected from the group consisting of: Q121S, Q121G, E129G, E129N, A130N, S132N, S132E, K133N, T134E, T134N, E142G, E142S, Y145S, Y146S, C178A, C178S, C178G, C194A, C194S, C194G, R200S, F201G, F201S, F201T, F201D, C218A, C218S, C218G, Q220E, Q220S, N240E, N240S, N240D, and N240T, and   the antibody Fc fragment (vi) is located C-terminally to the third CD40L domain (v), and is fused to the soluble CD40L domain (v) via a hinge-linker of any one of SEQ ID NOs: 16 and 19-24.   
     
     
         14 . The CD40 receptor agonist protein of  claim 13 , wherein the first and second peptide linkers (ii) and (iv) independently have a length of 3-8 amino acids. 
     
     
         15 . The CD40 receptor agonist protein of  claim 14 , wherein the first and the second peptide linkers (ii) and (iv) consist of the amino acid sequence of SEQ ID NO: 2. 
     
     
         16 . A pharmaceutical or diagnostic composition comprising the CD40 receptor agonist protein of  claim 13  and a pharmaceutically acceptable carrier.